Trehalose, a non-reducing disaccharide of glucose, is found at high concentrations in organisms that are capable of withstanding extreme drought and/or cold conditions in nature (i.e., anhydrobiosis or life without water). Moreover, trehalose has been demonstrated to be a potent, nontoxic bioprotectant for stabilizing lipids, proteins, viruses, blood cells and even eukaryotic mammalian cells (e.g., oocytes) at cryogenic and particularly, ambient temperatures (i.e., cryo and lyopreservation). Unfortunately, mammalian cells lack a mechanism to synthesize trehalose and the sugar cannot permeate their plasma membrane. However, trehalose must present both intra and extracellularly to protect cells from being damaged by the dehydration and/or freezing stresses during cryo and lyopreservation. Therefore, it is crucial to develop an effective approach that can deliver trehalose into mammalian cells as the first step toward long-term biostabilization of mammalian cells using the sugar, particularly at an ambient temperature. Due to the limited availability of cell sources, long-term cell biostabilization for future use is critical to the success of the emerging cell-based medical technologies such as tissue engineering, regenerative medicine, cell/organ transplantation, stem cell therapy, and assisted reproduction.
A number of methods have been explored to introduce trehalose within mammalian cells over the past two decades. The most straightforward approach is to deliver exogenous trehalose into the cytoplasm by direct microinjection. This approach has been successfully applied to oocytes that have a large size (˜100 μm in diameter) and are generally in a small quantity (tens or at most hundreds). However, it has difficulty to be applied to most mammalian cells that are generally much smaller (<20 μm) and in large quantities (usually millions). Mammalian cells have been genetically engineered to synthesize trehalose for biostabilization. This approach requires the constant production of adenoviral vectors at high multiplicities of infection that was found to exhibit significant cytotoxicity. Trehalose has also been introduced within mammalian cells or their organelles through engineered or natured transmembrane pores, electroporation, fluid-phase endocytosis, and lipid phase transition. More recently, liposomes have being investigated to encapsulate trehalose as a potential intracellular delivery vehicle of the sugar. However, consistent report of cryo and lyopreservation using trehalose delivered intracellularly via the above-mentioned approaches for small (<20 μm) eukaryotic mammalian cells, is still absent. This could be due to the inability to deliver a sufficient amount of intracellular trehalose (i.e., 0.1 M or more) for cellular protection using some of the approaches (e.g., fluid phase endocytosis). In addition, cells could be too severely compromised during the trehalose delivery steps to withstand further cryo/dehydration stress, considering the highly invasive nature of some of the approaches (e.g., electroporation).
Therefore, further investigation to develop a minimally invasive approach capable of delivering sufficient intracellular trehalose or similar agents for biostabilization is in need. Further, there is a more general need for efficient mechanisms for encapsulation and controlled release of small molecules for intracellular delivery.